The report of a single patient from Berlin who may be cured of his HIV disease almost 4 years after allogeneic stem cell transplant from a ?32 CCR5 donor has raised renewed excitement of the prospects for HIV gene therapy. One of the most promising vectors for doing so is based upon HIV, but current production methods are laborious and difficult to scale-up for possible clinical use. Here we propose the use of helper-dependent adenovirus or HDAd as a way to easily make HIV vector supernatants of high titer and quantity. In preliminary data we have made a single HDAd encoding all the HIV cis and trans components as a second generation vector. When introduced into 293 cells at high MOI resultant HIV titers were close to 108 IU/ml, 1-2 orders of magnitude greater than conventional multiplasmid transfection production methods. We were able to scale up production using a cell factory, and HIV vector was generated over several days. In this two-year proposal we now seek to extend this method to a third generation HIV vector. To do so, starting with an HDAd that already encodes VSV G, we will first incorporate an HIV packaging vector (PV) in several different sites and orientations. Once that testing is complete and the 1-2 best amplified HDAds are shown to be functional, an HIV transfer vector (TV) that encodes eGFP will be added, constructing up to a dozen HDAds that also have HIV-TV. After plasmid testing, the best 3-4 will be amplified, including one with all three HIV components. To produce HIV, 293-based cell lines will be transduced at increasing MOIs with the HDAds, supernatant harvested, and titered for HIV. HIV made from HDAd will be compared to that made using plasmid transfection in terms of pg CA per IU, genomic RNA content per IU, contamination with helper adenovirus, and ability to transduce non-dividing cells, including macrophages. Scalability will be shown using 3 l spinner flasks of suspension 293 cells. Finally, a TV encoding an anti-CCR5 shRNA will be incorporated into the HDAd and shown to be functional in transducing primary T cells and macrophages and protecting against M-tropic HIV challenge subsequently. At the end of the two year funding period we hope to have shown that it is possible to produce a fully functional, third generation VSV G-pseudotyped HIV vector using HDAd, of equal or greater titer compared to conventional plasmid transfection methods, which should have implications for the clinical use of these vectors for a wide range of congenital and acquired diseases, not merely HIV.